Use of sarp-1 for the treatment and/or prevention of scleroderma

ABSTRACT

The invention relates to the use of SARP-1 for the preparation of a medicament for the treatment and/or prevention of scleroderma, in particular of systemic sclerosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.10/432,256, which is a 371 national stage of PCT/EP01/13992, filed Nov.30, 2001, which claims priority from EP 00126771.5, filed Dec. 6, 2000,and EP 01118888.5, filed Aug. 17, 2001. The entire contents of priorapplications are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention is in the field of scleroderma. More specifically,the invention relates to the use of SARP-1 for the treatment and/orprevention of scleroderma, in particular of systemic sclerosis.

BACKGROUND OF THE INVENTION

Scleroderma is a disease of the connective tissue characterized byfibrosis of the skin and internal organs, leading to organ failure anddeath (Black et al., 1998; Clements and Furst, 1996). Scleroderma has aspectrum of manifestations and a variety of therapeutic implications. Itcomprises localized scleroderma, systemic sclerosis, scleroderma-likedisorders, and Sine scleroderma (Smith, 2000). Whilst localizedscleroderma is a rare dermatologic disease associated with fibrosis andmanifestations limited to skin, systemic sclerosis is a multisystemdisease with variable risk for internal organ involvement and variationin the extent of skin disease. Systemic sclerosis can be diffuse orlimited. Limited systemic sclerosis is also called CREST (calcinosis,Raynaud's esophageal dysfunction, sclerodaytyl), telangiectasiae).Scleroderma-like disorders are believed to be related to industrialenvironment exposure. In Sine disease, there is internal organinvolvement without skin changes.

The major manifestations of scleroderma and in particular of systemicsclerosis are inappropriate excessive collagen synthesis and deposition,endothelial dysfunction, spasm, collapse and obliteration by fibrosis.

Scleroderma is a rare disease with a stable incidence of approximately19 cases per 1 million persons. The cause of scleroderma is unknown.However, the genetic predisposition is important. Abnormalities involveautoimmunity and alteration of endothelial cell and fibroblast function.Indeed, systemic sclerosis is probably the most severe of theauto-immune diseases with 50% mortality within 5 years of diagnosis(Silman, 1991).

In terms of diagnosis, an important clinical parameter is skinthickening proximal to the metacarpophalangeal joints. Raynaud'sphenomenon is a frequent, almost universal component of scleroderma. Itis diagnosed by color changes of the skin upon cold exposure. Ischemiaand skin thickening are symptoms of Raynaud's disease.

Several underlying biological processes are implicated in theinitiation, severity and progression of the disease and include vasculardysfunction, endothelial cell activation and damage, leukocyteaccumulation, auto-antibody production and crucially, an uncontrolledfibrotic response which may lead to death (Clements and Furst, 1996).Fibroblasts have a pivotal role in the pathogenesis of this disease.Primary fibroblasts obtained from patients with scleroderma exhibit manyof the characteristic properties of the disease seen in vivo, notablyincreased extracellular matrix synthesis and deposition, notably ofcollagen and fibronectin, and altered growth factor and cytokineproduction such as of TGFβ and CTGF (Strehlow and Korn, 1998 and LeRoy,1974).

There is no curative treatment of scleroderma. Innovative but high-risktherapy proposed autologous stem cell transplantation (Martini et al.,1999). In particular, there are currently no treatments for sclerodermatargeting the fibrotic process (Wigley and Boling, 2000).

Identification of the genes associated with disease risk and sclerodermaprogression may lead to the development of effective strategies forintervention at various stages of the disease.

SARP-1 (secreted apoptosis-related protein 1) is a member of a family ofsecreted proteins known as secreted frizzled related proteins, based ontheir homology to the cysteine rich domain (CRD domain) found in thefrizzled family of 7 transmembrane receptors (Rattner et al., 1997).Frizzled genes were originally identified in drosophila and controltissue polarity (Adler et al., 1987). Frizzled proteins are thereceptors for the highly conserved Wnt family of at least 16 secretedsignaling molecules that regulate cell-to-cell interactions duringembryogenesis (Smalley and Dale, 1999). Insights into the mechanisms ofWnt action have emerged from several systems: genetics in Drosophila andC. elegans; biochemistry in cell culture; and ectopic gene expression inXenopus embryos. Many Wnt genes in the mouse have been mutated, leadingto very specific developmental defects. The Wnt signalling pathway whichis triggered by the interaction of Wnt with frizzled proteins, ismediated through several cytoplasmic relay components, and functions tosuppress the activity of the multiprotein β-catenin turnover complex,thus allowing a build up of cytosolic β-catenin which then enters thenucleus and forms a complex with TCF to activate transcription of Wnttarget genes (Miller et al., 1999; Kuhl et al., 2000).

Wnt-frizzled interactions may be modulated through the restrictedexpression of distinct Wnt binding proteins, the secreted frizzledrelated proteins (sFRP). SFRPs are able to bind Wnt through theN-terminal CRD domain. They can therefore sequester Wnt away from itsreceptors and thereby antagonize its effects (Bafico et al., 1999).

SARP-1 is known under several alternative names, such as SDF-5, PRO697,ATG-1622, HLHDY31, SFRP-2. Partial or full length protein and/or nucleicacid sequences of murine or human SARP-1 have been described in severalpatent applications, e.g. WO 98/35043, WO 98/13493, EP 0 879 887, WO99/46281.

In terms of function, the secreted frizzled-related proteins (SFRPs)appear to act as soluble modulators of Wnt signaling by competing withmembrane-bound frizzled receptors for the binding of secreted Wntligands. Apart from SARP-1, the human proteins of this family so farknown comprise SARP-2 (SFRP-1) and SARP-3 (SFRP-5). Murine SARP-1, andhuman SARPs-2 and -3 have been described to have the ability to eithersensitize cells to apoptosis or to inhibit the apoptotic response(Melkonyan et al., 1997). When expressed in a breast adenocarcinoma cellline, mouse SARP-1 and human SARP-2 exhibited opposite effects on cellsensitivity to pro-apoptotic stimuli. Whereas cells with SARP-1 hadhigher resistance, cells expressing SARP-2 were sensitized to apoptosisinduced by tumor necrosis factor and ceramide. Expression of SARP-1 orSARP-2 modified the intracellular levels of β-catenin, an indicator ofWnt mediated signal transduction, suggesting that SARPs interfere withthe Wnt-frizzled signaling pathway (Melkonyan et al., 1997).

Northern blot analysis revealed that the SARP genes have distinctexpression patterns (Leimeister et al., 1998). SARP-1 exists as 2.2- and1.3-kb transcripts in several human tissues, with the highest levels incolon and small intestine. Chang et al., 1999, reported that SARP-1, orSFRP-2, is highly and preferentially expressed in bovine retinathroughout the inner nuclear layer. Within the retina, SARP-3, orSFRP-5, is specifically expressed in the retinal pigment epithelium.

By analysis of somatic cell hybrids, Melkonyan et al. (1997) mapped theSARP-1 gene to human chromosome 4. Chang et al. (1999) refined the mapposition to 4q31.3 using radiation hybrid analysis.

There is also mounting evidence that an altered SARP-1 expression isrelated to cancer (WO 98/13493).

SUMMARY OF THE INVENTION

The invention is based on the finding of a beneficial effect of SARP-1in an established animal model of scleroderma.

It is therefore a first object of the invention to use SARP-1 for thepreparation of a medicament for the treatment and/or prevention ofscleroderma, and in particular of systemic sclerosis. It is a secondobject of the invention to use a cell expressing SARP-1 or an expressionvector comprising the coding sequence of SARP-1 for the preparation of amedicament for the treatment and/or prevention of scleroderma, inparticular systemic sclerosis. Pharmaceutical compositions comprisingSARP-1 and methods of treatment comprising administering SARP-1 to thehuman body are also within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression of SARP-1 mRNA in normal and diseasedfibroblasts from 7 scleroderma patients as determined by gene filteranalysis. The mean expression level for each class of samples (normal orabnormal) is given in (A) and the median is given in (B).

FIG. 2 shows the ratio of expression of SARP-1 mRNA in diseased/normalfibroblasts (low passage number, <5 passages) from 8 sclerodermapatients determined by real time PCR.

FIG. 3 shows the real time PCR analysis of SARP-1 mRNA expression innormal human dermal fibroblasts (NHDF) relative to GAPDH mRNAexpression.

FIG. 4 shows real time PCR analysis of SARP-1 mRNA expression inclinically normal human skin biopsies (n=6) compared to abnormal skinbiopsies from scleroderma patients (n=2) relative to GAPDH.

FIG. 5 A and B are contiguous and show the nucleotide sequence (SEQ IDNO:1) and deduced amino acid sequence (SEQ ID NO:2) of SARP-1 cDNA. Thepredicted cleavage positions for the signal peptide are indicated byarrows.

FIG. 6 shows the alignment of human (SEQ ID NO:2) and murine (SEQ IDNO:5) SARP-1 amino acid sequences. Non-identical residues between thetwo sequences are highlighted (gray).

FIG. 7 shows the level of apoptosis in SARP-1 transfected andmock-transfected cells compared to controls. *** is a statisticallysignificant reduction.

FIG. 8 shows the effect of SARP-1 treatment on total MMP-1 activity inconditioned medium harvested from human dermal fibroblast cultures. Thefibroblasts were derived from diseased tissue (A, D, F) or healthyindividuals (B, C), or from non-diseased areas of a scleroderma patient(E).

FIG. 9 shows the effect of SARP-1 cotransfection on collagen promoteractivity in unstimulated and TGFβ stimulated NIH3T3 cells. (A) indicatesthe luminescence units measured in the reporter assay, (B) gives thepercentage of luminescence with regard to the control.

FIG. 10 shows the fibosis score per lung in bleomycin-induced lungfibrosis model. The scores measured in mice having received vehicle,SARP-1-transfected cells, mock transfected cells or anti-TGF therapy isshown. *** is a statistically significant reduction.

DESCRIPTION OF THE INVENTION

In accordance with the present invention it has been found thatexpression of the secreted protein SARP-1 is significantly lower indiseased fibroblasts derived from scleroderma patients as compared tocontrol cells. DNA gene filter microarray technology has been used toidentify differentially expressed genes in skin fibroblasts isolatedfrom fibrotic lesions obtained from patients with scleroderma comparedto fibroblasts isolated from clinically unaffected areas of the skinfrom the same patients. The expression of SARP-1 was down regulated inthe lesional fibroblasts in 5 out of 7 patients tested. In addition tothis, real time RT-PCR analysis of total RNA isolated from whole biopsyspecimens of abnormal skin from scleroderma patients indicated lowerlevels of SARP-1 mRNA compared to clinically normal, age, sex andanatomical site matched control biopsies.

In support of the above results, a statistical method comparingexpression levels of all of the genes contained on the gene filtermicroarray indicated that the downregulation of SARP-1 had a 95%probability of being associated with the fibrotic lesions in thepatients, suggesting an association with the clinical progression of thedisease.

The beneficial effect of SARP-1 in scleroderma was confirmed in awell-established murine model of scleroderma, the bleomycin-induced lungfibrosis model. In this model, administration of SARP-1 expressing cellsreduced significantly the proportion of lung fibrosis.

Therefore, the invention relates to the use of a substance which bindsto and initiates signaling of the human SARP-1 receptor or a substancewhich stimulates release or potentiates the activity of endogenousSARP-1 for the manufacture of a medicament for the treatment and/orprevention of scleroderma. Said substance may be mature SARP-1 itself orany fragment of SARP-1 binding to and initiating signaling through theSARP-1 receptor, as well as any further agonist of the SARP-1 receptor,such as agonistic antibodies directed to the SARP-1 receptor or chemicalagonists specific therefor.

A receptor which has been suggested for SARP-1 is the wnt-protein,putatively binding to the cysteine rich frizzled (frz) domain of SARP-1(Lin et al., 1997). The substance according to the invention maytherefore also be a fragment of SARP-1 comprising the cysteine richfrizzled domain thereof.

Preferably, the substance used for treatment and/or prevention ofscleroderma is selected from the group consisting of:

(a) Mature SARP-1;

(b) A polypeptide comprising SEQ ID NO: 2;

(c) A polypeptide comprising amino acids 21 to 295 of SEQ ID NO: 2;

(d) A polypeptide comprising amino acids 24 to 295 of SEQ ID NO: 2;

(e) A polypeptide comprising amino acids 25 to 295 of SEQ ID NO: 2;

(f) A polypeptide comprising amino acids 26 to 295 of SEQ ID NO: 2;

(g) A polypeptide comprising amino acids 27 to 295 of SEQ ID NO: 2;

(h) A polypeptide comprising amino acids 28 to 295 of SEQ ID NO: 2;

(i) A polypeptide comprising amino acids 37 to 295 of SEQ ID NO: 2;

(j) A mutein of any of (a) to (i), wherein the amino acid sequence hasat least 40% or 50% or 60% or 70% or 80% or 90% identity to at least oneof the sequences in (a) to (i);

(k) A mutein of any of (a) to (i) which is encoded by a DNA sequencewhich hybridizes to the complement of the native DNA sequence encodingany of (a) to (i) under moderately stringent conditions or under highstringent conditions;

(l) A mutein of any of (a) to (i) wherein any changes in the amino acidsequence are conservative amino acid substitutions to the amino acidsequences in (a) to (i);

(m) a salt or an isoform, fused protein, functional derivative, activefraction or circularly permutated derivative of any of (a) to (l).

The full length cDNA of human SARP-1 has been cloned and is depicted inFIG. 5 as well as SEQ ID NO: 1 of the attached sequence listing. Thecorresponding amino acid sequence is given in FIG. 5 and SEQ ID NO: 2 ofthe attached sequence listing. As shown in the examples below, it hasbeen found that the N-terminus of SARP-1 is highly heterologous. Thesignal peptide of SARP-1 has been predicted to span either from aminoacids 25 to 295 of SEQ ID NO: 2 or from amino acids 21 to 295.N-terminal sequences of purified recombinant human SARP-1 expressed inHEK 293 cells has revealed further N-terminal sequences leading ofmature SARP-1 beginning at amino acids 24, 25, 26, 27, 28 or 37 of SEQID NO: 2.

The term “SARP-1”, as used herein, relates to any or all of thesubstances considered above in (a) to (m).

The term “treatment and/or prevention” as used herein encompasses anyattenuation, reduction, or partial, substantial or complete preventionor blockage of disease formation, development, progression or of theformation, development or progression of any one or several or all ofthe symptoms of the disease.

The term “scleroderma” as used herein comprises scleroderma in anyclassification and definition, as well as one or more of the symptoms ofscleroderma, as described in detail in the introduction. The term“scleroderma” further relates to the diseases known to be associatedwith scleroderma, such as the ones described in Smith (2000), forexample, which is fully incorporated by reference herein.

The term “scieroderma” as used herein comprises further fibroticdiseases such as liver cirrhosis, interstitial pulmonary fibrosis,Dupuytren's contracture, keloid and other scarring/wound healingabnormalities, postoperative adhesions and reactive fibrosis, as well aschronic heart failure, in particular after myocardial infarction. Theinvention relates to the use of SARP-1 for the manufacture of amedicament for the treatment and/or prevention of fibrotic diseases,such as the ones listed above.

Further diseases or disorders treatable with SAPR-1 comprise woundhealing diseases, in particular wound healing in the lung, comprisingchronic inflammation of the lung and ultimately fibrosis or scarring oflung surfaces. Disorders involving inflammation of the lung comprisee.g. idiopathic pulmonary fibrosis, sarcoidosis, bronchopulmonarydysplasia, fibroproliferative ARDS, as well as pulmonary manifestationsor systemic diseases such as rheumatoid arthritis (Krein et al., 2001).

The term “scleroderma” preferably relates to localized, systemic,limited and diffuse scleroderma as well as overlap syndromes.

Localized scleroderma primarily affects the skin, but may also affectthe underlying muscles and bones. However, it does not affect internalorgans. Localized scleroderma is relatively mild, and may be related tosystemic scleroderma in terms of similar superficial symptoms, such asthe appearance of skin biopsy under the microscope.

Systemic scleroderma comprises several types of symptoms or groups ofsymptoms, such as CREST, limited and diffuse. Systemic scleroderma isalso known as systemic sclerosis. It may also be referred to asprogressive systemic sclerosis, or familial progressive systemicsclerosis. Systemic scleroderma may e.g. affect the skin, blood vessels,and/or internal organs. When it affects the skin, it can cause the skinto harden, most commonly on the hands and/or face. When it affects theblood vessels, it can cause Raynaud's disease. The most serious forms ofsystemic sclerosis affect the internal organs, and may cause disabilityor even death. Among others, systemic sclerosis comprises: sclerodermalung disease, scleroderma renal crisis, cardiac manifestations, muscularweakness including fatigue or limited CREST, gastrointestinaldysmotility and spasm, and abnormalities in the central, peripheral andautonomic nervous system. With regard to the nervous systemabnormalities, carpal tunnel syndrome followed by trigeminal neuralgiaare the most common.

Limited Scleroderma may be limited to the hands, although the face andneck may also be involved.

Diffuse Scleroderma comprises skin tightening and also occurs above thewrists (or elbows). There are several subcategories of diffuse systemicsclerosis, such as “scleroderma sine scleroderma” where there isinternal organ fibrosis, but no skin tightening; and familialprogressive systemic sclerosis, a rare form which occurs in families.

Overlap syndromes are referred to if a scleroderma patient also hasother autoimmune disease (such as lupus, rheumatoid arthritis, etc.), ase.g. in diffuse scleroderma in overlap with lupus. Scleroderma symptomscan also be a part of mixed connective tissue disease (MCTD), orUndifferentiated Connective Tissue Disease (UCTD).

The term “SARP-1” as used herein, relates to a protein comprising all ora portion of the sequence of SEQ ID NO: 2 (human) or SEQ ID NO: 5(murine) of the enclosed sequence listing, irrespective of thedesignation of such protein, including, but not limited to the furtherknown designations SDF-5, PRO697, ATG-1622, HLHDY31, SFRP-2 or FRP-2, aswell as to salts, isoforms, muteins, active fractions, functionalderivatives and circularly permutated derivatives thereof.

Preferably, the term “SARP-1” refers to a mature protein lacking thesignal peptide. The signal peptide is predicted to contain the first 20or the first 23, 24, 25, 26, 27 or 36 amino acids of SAPP-1 as definedin SEQ ID NO: 2, meaning that the mature protein would either compriseamino acids 21 to 295 or 24, 25, 26, 27, 28 or 37 to 295 of SEQ ID NO:2.

As shown in FIG. 6 below, the human amino acid sequence of SARP-1 ishighly homologous to the murine sequence (SEQ ID NO: 5). Therefore,murine SARP-1 may also be used according to the invention, as well asproteins derived from other species, as long as there is a sufficientlyhigh identity between the proteins as to allow the protein to exhibitits biological activity, and without eliciting a substantial immuneresponse in a human being.

The term “SARP-1” further relates to any fragment, portion, domain orsub-domain of SEQ ID NO: 2 or 5 showing the desired activity inscleroderma. Protein fragments or one or more domains of the protein maybe used according to the invention, as long as they exhibit anybeneficial effect on scleroderma, preferable an effect which is at leastcomparable of the full length protein. The beneficial effect can bemeasured in one of the in vitro or in vivo tests described in theexamples below, or in any other assay adequate to demonstrate an effectin scleroderma. For example, SARP-1 comprises a cysteine rich frizzleddomain, and a netrin-like domain, also called NTR module, which ishomologous to tissue inhibitors of metalloproteinases (TIMPs) (Banyaiand Patthy, 1999). Therefore, a fragment comprising the frizzled domainof SARP-1, or a fragment comprising the NTR module are preferredfragments according to the invention.

In accordance with the present invention, SARP-1 can be a naturallyoccurring, i.e. native protein, or a recombinant protein. Recombinantproduction may be carried out in eukaryotic cells, such as yeast cellsor CHO cells, in human fibroblast cells or cell lines. It may further beproduced in prokaryotic cells such as E. coli.

Preferably, SARP-1 is glycosylated at one or more sites. It may also beunglycosylated, depending on the given needs and the source ofproduction or isolation of the protein.

The term “salts” herein refers to both salts of carboxyl groups and toacid addition salts of amino groups of SARP-1 molecule or analogsthereof. Salts of a carboxyl group may be formed by means known in theart and include inorganic salts, for example, sodium, calcium, ammonium,ferric or zinc salts, and the like, and salts with organic bases asthose formed, for example, with amines, such as triethanolamine,arginine or lysine, piperidine, procaine and the like. Acid additionsalts include, for example, salts with mineral acids, such as, forexample, hydrochloric acid or sulfuric acid, and salts with organicacids, such as, for example, acetic acid or oxalic acid. Of course, anysuch salts must retain the biological activity of SARP-1 relevant to thepresent invention, i.e., exert a beneficial effect on scleroderma.

Isoforms or splice variants of SARP-1 may also be used according to theinvention, as long as they are capable of inhibiting scleroderma diseaseprogression and/or symptoms of that disease. The two 1.3 kb and 2.2 kbtranscripts found to be differentially expressed in human tissues mayfor instance represent different isoforms of SARP-1, which could beuseful according to the invention.

As used herein the term “muteins” refers to analogs of an SARP-1, inwhich one or more of the amino acid residues of a natural SARP-1 arereplaced by different amino acid residues, or are deleted, or one ormore amino acid residues are added to the natural sequence of SARP-1,having preferably at least the same activity as wild type SARP-1 or evenhaving a much more potent activity. These muteins are prepared by knownsynthesis and/or by site-directed mutagenesis techniques, or any otherknown technique suitable therefor.

Any such mutein preferably has a sequence of amino acids sufficientlyduplicative of that of SARP-1, as described in SEQ ID NO: 2, such as tohave at least a substantially similar activity of SARP-1. The activityof a SARP-1 mutant can be tested by assays know n in the art, and inparticular using the assays explained in the examples below. Measuringthe amount of collagen synthesis (example 7) in fibroblasts is asuitable test for assessing the activity of SARP-1 muteins, for example.

Muteins in accordance with the present invention include proteinsencoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNAor RNA, which encodes SARP-1, in accordance with the present invention,under stringent conditions. The term “stringent conditions” refers tohybridization and subsequent washing conditions, which those of ordinaryskill in the art conventionally refer to as “stringent”. See Ausubel etal., Current Protocols in Molecular Biology, supra, Interscience, N.Y.,§§6.3 and 6.4 (1987, 1992), and Sambrook et al. (Sambrook, J. C.,Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Without limitation, examples of stringent conditions include washingconditions 12-20° C. below the calculated Tm of the hybrid under studyin, e.g., 2×SSC and 0.5% SDS for 5 minutes, 2×SSC and 0.1% SDS for 15minutes; 0.1×SSC and 0.5% SDS at 37° C. for 30-60 minutes and then, a0.1×SSC and 0.5% SDS at 68° C. for 30-60 minutes. Those of ordinaryskill in this art understand that stringency conditions also depend onthe length of the DNA sequences, oligonucleotide probes (such as 10-40bases) or mixed oligonucleotide probes. If mixed probes are used, it ispreferable to use tetramethyl ammonium chloride (TMAC) instead of SSC.See Ausubel, supra.

Any such mutein preferably has a sequence of amino acids sufficientlyduplicative of that of SARP-1, such as to have substantially similar, oreven better, biological activity as SARP-1.

One easily measurable activity of SARP-1 is its capability of reducingcollagen synthesis. An assay for measuring this activity is described indetail in example 6 below. As long as the mutein has substantialcollagen reducing activity, it can be considered to have substantiallysimilar activity to SARP-1. Thus, it can be determined whether any givenmutein has at least substantially the same activity as SARP-1 by meansof routine experimentation comprising subjecting such a mutein, e.g., toa simple assay as described in example 7.

In a preferred embodiment, any such mutein has at least 40% identity orhomology with the sequence of SEQ ID NO: 2 of the annexed sequencelisting. More preferably, it has at least 50%, at least 60%, at least70%, at least 80% or, most preferably, at least 90% identity or homologythereto.

Identity reflects a relationship between two or more polypeptidesequences or two or more polynucleotide sequences, determined bycomparing the sequences. In general, identity refers to an exactnucleotide to nucleotide or amino acid to amino acid correspondence ofthe two polynucleotides or two polypeptide sequences, respectively, overthe length of the sequences being compared.

For sequences where there is not an exact correspondence, a “% identity”may be determined. In general, the two sequences to be compared arealigned to give a maximum correlation between the sequences. This mayinclude inserting “gaps” in either one or both sequences, to enhance thedegree of alignment. A % identity may be determined over the wholelength of each of the sequences being compared (so-called globalalignment), that is particularly suitable for sequences of the same orvery similar length, or over shorter, defined lengths (so-called localalignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequencesare well known in the art. Thus for instance, programs available in theWisconsin Sequence Analysis Package, version 9.1 (Devereux J et al1984), for example the programs BESTFIT and GAP, may be used todetermine the % identity between two polynucleotides and the % identityand the % homology between two polypeptide sequences. BESTFIT uses the“local homology” algorithm of Smith and Waterman (1981) and finds thebest single region of similarity between two sequences. Other programsfor determining identity and/or similarity between sequences are alsoknown in the art, for instance the BLAST family of programs (Altschul SF et al, 1990, Altschul S F et al, 1997, accessible through the homepage of the NCBI at www.ncbi.nim.nih.gov) and FASTA (Pearson W R, 1990;Pearson 1988).

Muteins of SARP-1, which can be used in accordance with the presentinvention, or nucleic acids coding therefor, include a finite set ofsubstantially corresponding sequences as substitution peptides orpolynucleotides which can be routinely obtained by one of ordinary skillin the art, without undue experimentation, based on the teachings andguidance presented herein.

Preferred changes for muteins in accordance with the present inventionare what are known as “conservative” substitutions. Conservative aminoacid substitutions of SARP-1 polypeptides or proteins, may includesynonymous amino acids within a group which have sufficiently similarphysicochemical properties that substitution between members of thegroup will preserve the biological function of the molecule (Grantham,1974). It is clear that insertions and deletions of amino acids may alsobe made in the above-defined sequences without altering their function,particularly if the insertions or deletions only involve a few aminoacids, e.g., under thirty, and preferably under ten, and do not removeor displace amino acids which are critical to a functional conformation,e.g., cysteine residues. Proteins and muteins produced by such deletionsand/or insertions come within the purview of the present invention.

Preferably, the synonymous amino acid groups are those defined inTable 1. More preferably, the synonymous amino acid groups are thosedefined in Table II; and most preferably the synonymous amino acidgroups are those defined in Table III. TABLE I Preferred Groups ofSynonymous Amino Acids Amino Acid Synonymous Group Ser Ser, Thr, Gly,Asn Arg Arg, Gln, Lys, Glu, His Leu Ile, Phe, Tyr, Met, Val, Leu ProGly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His, Gln, Thr Ala Gly, Thr,Pro, Ala Val Met, Tyr, Phe, Ile, Leu, Val Gly Ala, Thr, Pro, Ser, GlyIle Met, Tyr, Phe, Val, Leu, Ile Phe Trp, Met, Tyr, Ile, Val, Leu, PheTyr Trp, Met, Phe, Ile, Val, Leu, Tyr Cys Ser, Thr, Cys His Glu, Lys,Gln, Thr, Arg, His Gln Glu, Lys, Asn, His, Thr, Arg, Gln Asn Gln, Asp,Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu, Asn, Asp Glu Asp, Lys,Asn, Gln, His, Arg, Glu Met Phe, Ile, Val, Leu, Met Trp Trp

TABLE II More Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg His, Lys, Arg Leu Leu, Ile, Phe, Met ProAla, Pro Thr Thr Ala Pro, Ala Val Val, Met, Ile Gly Gly Ile Ile, Met,Phe, Val, Leu Phe Met, Tyr, Ile, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser HisHis, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, AsnGlu Glu, Gln Met Met, Phe, Ile, Val, Leu Trp Trp

TABLE III Most Preferred Groups of Synonymous Amino Acids Amino AcidSynonymous Group Ser Ser Arg Arg Leu Leu, Ile, Met Pro Pro Thr Thr AlaAla Val Val Gly Gly Ile Ile, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser HisHis Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Met, Ile, Leu Trp Met

Examples of production of amino acid substitutions in proteins which canbe used for obtaining muteins of SARP-1 polypeptides or proteins, foruse in the present invention include any known method steps, such aspresented in U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, to Market al; U.S. Pat. No. 5,116,943 to Koths et al., U.S. Pat. No. 4,965,195to Namen et al; U.S. Pat. No. 4,879,111 to Chong et al; and U.S. Pat.No. 5,017,691 to Lee et al; and lysine substituted proteins presented inU.S. Pat. No. 4,904,584 (Shaw et al).

The term “fused protein” refers to a polypeptide comprising SARP-1, or amutein thereof, fused with another protein, which, e.g., has an extendedresidence time in body fluids. Fusion proteins comprising all or afunctional part of SARP-1 fused to all or a functional part of a proteincapable of improving the biological activities of the molecule, likehalf-life in the human body, for instance, are preferred according tothe invention. In a preferred embodiment the fused protein comprises animmunoglobulin (Ig) fusion. Fusion proteins comprising all or part ofSARP-1 fused to all or part of an immunoglobulin are highly preferred.They can be monomeric or multimeric, hetero- or homomultimeric.Advantageously, the fused protein comprises the constant region of animmunoglobulin, in particular of the Fc portion of the immunoglobulin.Embodiments in which the immunoglobulin is of the IgG1 or IgG2 isotypeare further preferred according to the invention.

SARP-1 may thus be fused to another protein, polypeptide or the like,e.g., an immunoglobulin or a fragment thereof. The fusion may be direct,or via a short linker peptide which can be as short as 1 to 3 amino acidresidues in length or longer, for example, 13 amino acid residues inlength. Said linker may be a tripeptide of the sequence E-F-M(Glu-Phe-Met), for example, or a 13-amino acid linker sequencecomprising Glu-Phe-Gly-Ala-Gly-Leu-Val-Leu-Gly-Gly-Gln-Phe-Met (SEQ IDNO:6) introduced between the SARP-1 sequence and the immunoglobulinsequence.

“Functional derivatives” as used herein cover derivatives of SARP-1, andtheir muteins and fused proteins, which may be prepared from thefunctional groups which occur as side chains on the residues or the N-or C-terminal groups, by means known in the art, and are included in theinvention as long as they remain pharmaceutically acceptable, i.e. theydo not destroy the activity of the protein which is at leastsubstantially similar to the activity of SARP-1, and do not confer toxicproperties on compositions containing it. Therefore, in a preferredembodiment the functional derivative comprises at least one moietyattached to one or more functional groups which occur as one or moreside chains on the amino acid residues.

In accordance with the present invention, polyethylene glycol (PEG)side-chains are highly preferred moieties. PEG side chains may maskantigenic sites and extend the residence of the substance they areattached to in body fluids. Other derivatives include aliphatic estersof the carboxyl groups, amides of the carboxyl groups by reaction withammonia or with primary or secondary amines, N-acyl derivatives of freeamino groups of the amino acid residues formed with acyl moieties (e.g.alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of freehydroxyl groups (for example that of seryl or threonyl residues) formedwith acyl moieties.

“Active fractions” of SARP-1 and its muteins and fused proteins, coverany fragment or precursors of the polypeptide chain of the proteinmolecule alone or together with associated molecules or residues linkedthereto, e.g., sugar or phosphate residues, or aggregates of the proteinmolecule or the sugar residues by themselves, provided said activefraction has at least a substantially similar activity to SARP-1.

The invention further relates to the use of a nucleic acid molecule formanufacture of a medicament for the treatment and/or prevention ofscleroderma, wherein the nucleic acid molecule comprises a nucleic acidsequence encoding a polypeptide comprising an amino acid sequenceselected from the group consisting of:

(a) Mature SARP-1;

(b) A polypeptide comprising SEQ ID NO: 2;

(c) A polypeptide comprising amino acids 21 to 295 of SEQ ID NO: 2;

(d) A polypeptide comprising amino acids 24 to 295 of SEQ ID NO: 2;

(e) A polypeptide comprising amino acids 25 to 295 of SEQ ID NO: 2;

(f) A polypeptide comprising amino acids 26 to 295 of SEQ ID NO: 2;

(g) A polypeptide comprising amino acids 27 to 295 of SEQ ID NO: 2;

(h) A polypeptide comprising amino acids 28 to 295 of SEQ ID NO: 2;

(i) A polypeptide comprising amino acids 37 to 295 of SEQ ID NO: 2;

(j) A mutein of any of (a) to (i), wherein the amino acid sequence hasat least 40% or 50% or 60% or 70% or 80% or 90% identity to at least oneof the sequences in (a) to (i);

(k) A mutein of any of (a) to (i) which is encoded by a DNA sequencewhich hybridizes to the complement of the native DNA sequence encodingany of (a) to (i) under moderately stringent conditions or under highstringent conditions;

(l) A mutein of any of (a) to (i) wherein any changes in the amino acidsequence are conservative amino acid substitutions to the amino acidsequences in (a) to (i);

(m) a salt or an isoform, fused protein, functional derivative, activefraction or circularly permutated derivative of any of (a) to (l)

for the manufacture of a medicament for the treatment and/or preventionof scleroderma. The invention equally relates to the use of said nucleicacid molecules for the treatment and/or prevention of scleroderma.

In accordance with the present invention, SARP-1 may also beadministered to the human body in form of a vector comprising saidnucleic acid molecule. Therefore, the invention further relates to theuse of a vector comprising said nucleic acid molecule for themanufacture of a medicament for the treatment and/or prevention ofscleroderma. Preferably, the vector is an expression vector, comprisinga promoter operably linked to all or part of the coding sequence ofSARP-1. In a further preferred embodiment, the vector is a gene therapyvector. Gene therapy vectors are known in the art, most of them arevirally derived vectors, such as adenoviral or lentiviral vectors.

According to the invention, SARP-1 may also be administered to the humanbody in form of a cell producing and/or secreting SARP-1. Therefore, theinvention further relates to the use of a cell expressing SARP-1 for themanufacture of a medicament for the treatment and/or prevention ofscleroderma, i.e. to cell therapy for the treatment and/or prevention ofscleroderma. The cell may be a naturally producing SARP-1 and/or atransfected cell that produces recombinant SARP-1. Preferred are cellsexpressing and secreting high amounts of the protein, such asover-expressing cells carrying high copy numbers of an expression vectorcomprising a nucleic acid molecule encoding SARP-1.

As fibroblasts represent the machinery of fibrosis they are the mostsuitable cells for anti-fibrotic and scleroderma therapy. Therefore,preferably, SARP-1 expressing fibroblasts are used in accordance withthe present invention.

The invention further relates to a cell comprising a vector comprising anucleic acid molecule encoding all or part of SARP-1 for the preparationof a medicament for treatment and/or prevention of scleroderma. A cellthat has been genetically modified to produce a polypeptide according tothe invention is also within the scope of the present invention.

The use of an expression vector for inducing and/or enhancing theendogenous production of SARP-1 in a cell normally silent or expressingamounts of the inhibitor which are not sufficient, are also contemplatedaccording to the invention. Thus, the invention makes use of atechnology known as endogenous gene activation (EGA) for the productionof the desired protein.

Systemic sclerosis is one of the most serious diseases withinscleroderma, often leading to disablement and death. Therefore, afurther preferred embodiment of the invention relates to systemicsclerosis, which is an indication within the disease of scleroderma,being characterized mainly by involvement of internal organs, asdescribed in detail above.

Several combination treatments are preferred in accordance with thepresent invention. Therefore, preferably, the medicament of theinvention further comprises:

-   -   Interferon, in particular interferon-β    -   A Tumor Necrosis Factor (TNF) antagonist, in particular TBPI        and/or TBP II    -   A further anti-scleroderma agent    -   An anti-scleroderma agent selected from the group consisting of        ACE inhibitors, calcium channel blockers, proton pump        inhibitors, NSAIDs, COX-inhibitors, corticosteroids,        tetracycline, pentoxifylline, bucillamine, geranylgeranyl        transferase inhibitors, rotterlin, prolyl-4-hydroxlase        inhibitors, c-proteinase inhibitors, lysyl-oxidase inhibitors,        relaxin, halofuginone, prostaglandins, prostacyclins,        endothelin-1, nitric oxide, angiotensin II inhibitors and        anti-oxidants.

All treatments are intended for simultaneous, sequential or separateuse.

Although there is presently no cure for scleroderma, several agents ortreatments are presently being used to treat scleroderma symptoms. Suchanti-scleroderma agents, which may be used as combination therapyaccording to the invention, are summarized e.g. in Leighton (2001) orWigley and Sule (2001), which are fully incorporated by referenceherein.

Interferons are predominantly known for inhibitory effects on viralreplication and cellular proliferation. Interferon-y, for example, playsan important role in promoting immune and inflammatory responses.Interferon D (IFN-β, an interferon type 1), is said to play ananti-inflammatory role.

In yet a further embodiment of the invention, SARP-1 is used incombination with a TNF antagonist. TNF antagonists exert their activityin several ways. First, antagonists can bind to or sequester the TNFmolecule itself with sufficient affinity and specificity to partially orsubstantially neutralise the TNF epitope or epitopes responsible for TNFreceptor binding (hereinafter termed “sequestering antagonists”). Asequestering antagonist may be, for example, an antibody directedagainst TNF.

Alternatively, TNF antagonists can inhibit the TNF signalling pathwayactivated by the cell surface receptor after TNF binding (hereinaftertermed “signalling antagonists”). TNF antagonists are easily identifiedand evaluated by routine screening of candidates for their effect on theactivity of native TNF on susceptible cell lines in vitro, for examplehuman B cells, in which TNF causes proliferation and immunoglobulinsecretion. The assay contains TNF formulation at varying dilutions ofcandidate antagonist, e.g. from 0.1 to 100 times the molar amount of TNFused in the assay, and controls with no TNF or only antagonist (Tucci etal., 1992).

Sequestering antagonists are the preferred TNF antagonists to be usedaccording to the present invention. Amongst sequestering antagonists,those polypeptides that bind TNF with high affinity and possess lowimmunogenicity are preferred. Soluble TNF receptor molecules andneutralising antibodies to TNF are particularly preferred. For example,soluble forms of TNF-RI (p55) and TNF-RII (p75) are useful in thepresent invention. Truncated forms of these receptors, comprising theextracellular domains of the receptors or functional portions thereof,are more particularly preferred antagonists according to the presentinvention. Truncated soluble TNF type-I and type-II receptors aredescribed in EP914431, for example.

Truncated forms of the TNF receptors are soluble and have been detectedin urine and serum as about 30 kDa or 40 kDa TNF inhibitory bindingproteins, which are called TBPI and TBPII, respectively (Engelmann etal., 1990). The simultaneous, sequential, or separate use of SARP-1 withthe TNF antagonist and/or an Interferon is preferred, according to theinvention.

According to the invention, TBPI and TBPII are preferred TNF antagoniststo be used in combination with an SARP-1. Derivatives, fragments,regions and biologically active portions of the receptor moleculesfunctionally resemble the receptor molecules that can also be used inthe present invention. Such biologically active equivalent or derivativeof the receptor molecule refers to the portion of the polypeptide, or ofthe sequence encoding the receptor molecule, that is of sufficient sizeand able to bind TNF with such an affinity that the interaction with themembrane-bound TNF receptor is inhibited or blocked.

In a further preferred embodiment, human soluble TNF-RI (TBPI) is theTNF antagonist to be used according to the invention. The natural andrecombinant soluble TNF receptor molecules and methods of theirproduction have been described in the European Patents EP 308 378, EP398 327 and EP 433 900.

Whilst it may be beneficial to block TNF-α in early stages of thedisease, it has been discussed that in later stages, TNF itself mayexert a beneficial effect on scleroderma (Abraham et al., 2000).Therefore, the invention further relates to a combination of SARP-1 andTNF-α for treatment or prevention of scleroderma, in particular inadvanced stages of disease.

COX inhibitors are known in the art. Specific COX-2 inhibitors aredisclosed in WO 01/00229, for example.

The invention further relates to a pharmaceutical composition comprisingSARP-1, optionally together with one or more pharmaceutically acceptablecarriers, diluents or excipients, for the treatment and/or prevention ofscleroderma, in particular systemic sclerosis. The pharmaceuticalcomposition may further comprise any of the above-identified furthercomponents, and in particular an interferon, a TBP or a COX inhibitor.

The pharmaceutical composition according to the invention may alsocomprise a vector comprising a nucleic acid molecule according to theinvention, or a cell expressing SARP-1.

The active ingredients of the pharmaceutical, i.e. polypeptides, nucleicacids or cells according to the invention, or combinations thereof, aswell as the combinations of substances mentioned above, may beadministered to an individual in a variety of ways. The routes ofadministration include intradermal, transdermal (e.g. in slow releaseformulations), intramuscular, intraperitoneal, intravenous,subcutaneous, oral, epidural, topical, and intranasal routes. Any othertherapeutically efficacious route of administration can be used, forexample absorption through epithelial or endothelial tissues or by genetherapy wherein a DNA molecule encoding the active agent is administeredto the patient (e.g. via a vector) which causes the active agent to beexpressed and secreted in vivo. In addition, the protein(s) according tothe invention can be administered together with other components ofbiologically active agents such as pharmaceutically acceptablesurfactants, excipients, carriers, diluents and vehicles.

The definition of “pharmaceutically acceptable” is meant to encompassany carrier, which does not interfere with effectiveness of thebiological activity of the active ingredient and that is not toxic tothe host to which it is administered. For example, for parenteraladministration, the active protein(s) may be formulated in a unit dosageform for injection in vehicles such as saline, dextrose solution, serumalbumin and Ringers solution.

For parenteral (e.g. intravenous, subcutaneous, intramuscular)administration, the active protein(s) can be formulated as a solution,suspension, emulsion or lyophilised powder in association with apharmaceutically acceptable parenteral vehicle (e.g. water, saline,dextrose solution) and additives that maintain isotonicity (e.g.mannitol) or chemical stability (e.g. preservatives and buffers). Theformulation is sterilized by commonly used techniques.

The bioavailability of the active protein(s) according to the inventioncan also be ameliorated by using conjugation procedures which increasethe half-life of the molecule in the human body, for example linking themolecule to polyethylenglycol, as described in the PCT PatentApplication WO 92/13095.

The therapeutically effective amount of the active protein(s) will be afunction of many variables, including the type of receptor, the affinityof the substance according to the invention to its receptor, anyresidual cytotoxic activity exhibited thereby, the route ofadministration, the clinical condition of the patient.

A “therapeutically effective amount” is such that when administered, thesubstance according to the invention results in activating the SARP-1receptor, or inactivating a ligand stimulating the receptor in vivo. Thedosage administered, as single or multiple doses, to an individual willvary depending upon a variety of factors, including SARP-1pharmacokinetic properties, the route of administration, patientconditions and characteristics (sex, age, body weight, health, size),extent of symptoms, concurrent treatments, frequency of treatment andthe effect desired. Adjustment and manipulation of established dosageranges are well within the ability of those skilled in the art.

The dose of the polypeptide according to the invention required willvary from about 0.0001 to 100 mg/kg or about 0.01 to 10 mg/kg or about0.1 to 5 mg/kg or about 1 to 3 mg/kg, although as noted above this willbe subject to a great deal of therapeutic discretion.

The daily doses are usually given in divided doses or in sustainedrelease form effective to obtain the desired results. Second orsubsequent administrations can be performed at a dosage, which is thesame, less than or greater than the initial or previous doseadministered to the individual. A second or subsequent administrationcan be administered during or prior to onset of the disease.

The invention further relates to a method for treating and/or preventingscleroderma, in particular systemic sclerosis, comprising administeringto a patient in need thereof an effective amount of a polypeptideaccording to the invention, optionally together with a pharmaceuticallyacceptable carrier. Alternatively, or additionally, a cell producingSARP-1 or a nucleic acid molecule of the invention, optionally comprisedin an expression vector, may be administered according to the invention.

The expression vector may be administered systemically. Preferably theexpression vector is administered by intramuscular injection. A furtherpreferred route of administration is inhalation, in particular if lungfibrosis is involved in the disease.

The invention further relates to a method for the preparation of apharmaceutical composition comprising admixing an effective amount ofSARP-1 with a pharmaceutically acceptable carrier, and to a method oftreatment and/or prevention of arthritis comprising administering to ahost in need thereof an effective inhibiting amount of SARP-1.

All references cited herein, including journal articles or abstracts,published or unpublished U.S. or foreign patent application, issued U.S.or foreign patents or any other references, are entirely incorporated byreference herein, including all data, tables, figures and text presentedin the cited references. Additionally, the entire contents of thereferences cited within the references cited herein are also entirelyincorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplication such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning an range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

Having now described the invention, it will be more readily understoodby reference to the following examples that are provided by way ofillustration and are not intended to be limiting of the presentinvention.

EXAMPLES Example 1 SARP-1 is Differentially Expressed in SkinFibroblasts from Scleroderma Patients

Methods

Patient Samples

Two mm3 punch biopsies were taken from lesional or non-lesional skin(usually from forearm skin) of nine age and sex matched patients withdiffuse cutaneous systemic sclerosis (SSc). All patients fulfilled thecriteria of the American College of Rheumatology for the diagnosis ofsystemic sclerosis.

Fibroblast cultures.

Fibroblasts were obtained from the biopsies by in vitro culture aspreviously described (Abraham et al., 1991). Briefly, biopsies were cutinto pieces and placed in sterile plastic dishes or flasks. After 15minutes of drying at room temperature the pieces of biopsy were adherentto the tissue culture plastic and were then cultured in fibroblastgrowth medium (FGM) consisting of Dulbecco's modified Eagle's medium(DMEM) containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 1 mMsodium pyruvate, 100 units per ml penicillin, 100 μg per mlstreptomycin, 50 μg per ml gentamicin and 2.5 μg per ml amphotericin B.After 2-3 weeks of incubation in a humidified atmosphere of 5% CO₂ inair, the fibroblast outgrowths were detached by brief trypsin treatmentand re-cultured in FGM without gentamicin and amphotericin B. Inexperiments, fibroblasts were used between passages 2 and 5. Thefibroblast phenotype was confirmed by their typical morphology inmonolayer and three-dimensional collagen gel cultures.

RNA Isolation

Total RNA was isolated from confluent scleroderma fibroblasts in earlypassage or from primary cultures (passage 1-3) of normal human dermal(foreskin) fibroblasts (purchased from Promocell) using Trizol (LifeTechnologies) according to the manufacturer's protocol. The final RNApellet was resuspended in sterile DEPC treated water at a concentrationof 1 μg/μl and stored at −80 C. cDNA probe synthesis 2 μg total RNA wasmixed with 1.3 μl of cytokine specific primers (R&D systems cat. no.GAC11) and incubated at 70 C for 2 min a 0.5 ml eppendorf tube. Thetubes were then cooled to 50 C for 2 min after which time reactionmixture containing 2 μl of 5× reaction buffer (250 mM Tris-HCl pH 8.3,375 mM KCl and 15 mM MgCl₂), 1 μl of 10× dNTP mixture (5 mM dGTP, 5 mMdCTP and 5 mM dTTP), 3.5 μl of α32P-dATP (3000 Ci/mmol, Amersham cat.no. PB10204), 0.5 μl DTT (100 mM) and 1 μl of Superscript II (LifeTechnologies) were added. The reaction mixture was mixed briefly bypipetting and incubated at 50 C for 25 min. The reaction was stopped byaddition of 1 μl of 0.1 M EDTA pH 8.0 containing 1 mg/ml glycogen). Thelabelled cDNA was then purified from un-incorporated deoxynucleotidesusing a Chromaspin-200 DEPC-H20 column (Clontech) according to themanufacturer's instructions. cDNA containing fractions were identifiedby Czerenkov counting. The peak fraction (normally fraction 2 out of atotal of 6 collected) was treated with 0.1 volumes of 1 M NaOHcontaining 1 mM EDTA for 20 min at 70 C to hydrolyze the RNA, and thenneutralized with an equal volume of 1 M NaH2PO4 containing 2.5 μg ofhuman Cot-1 DNA (Life Technologies) for 20 min at 70 C. The heat treatedneutralized cDNA probe was then added directly to the hybridizationmixture.

Hybridization to Gene Filter Microarrays

Gene filter microarrays (human cytokine expression array, R&D systemscat no. GA001) were prehybridized at 68 C for 2 h in 5 ml of ExpressHybridization solution (Clontech) containing 0.5 μg/ml salmon sperm DNA(Life technologies) in roller bottles, in a Hybaid hybridization oven(MWG Biotech). After this time the prehybrization solution was replacedwith fresh hybridization solution containing the cDNA probe preparationat a specific activity of between 0.25 to 1×106 cpm/ml. Hybridizationwas for 16-20 h at 68 C. After hybridization, filters were washed 4times with 2×SSC/1% SDS at 68 C for 20 minutes per wash and twice with0.1×SSC/0.5% SDS for 15 min per wash. Filters were then sealed in Saranwrap™ and exposed to a K-type storage phosphor imaging screen (Biorad)for varying times (4 h to 4 days) at room temperature.

Image Analysis

Imaging screens were scanned at a resolution of 50 μm using a BioradPersonal FX phosphoimager. The resultant 16 bit digital file wasconverted to TIF format and the image analyzed using Arrayvisionsoftware (Imaging Research Inc.). For each sample, we measured the pixelintensity of the 384 genes spotted in duplicate on the filter. Thebackground signal was subtracted and an average pixel intensity for eachpair of spots in the array was generated (=expression level).

Confirmation of microarray results on selected genes by RT-PCR.

1 μg of total RNA from each patient sample was reverse transcribed usingan oligo dT primer (Promega) in a 20 μl reaction volume containing 5 mMMgCl₂, 10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100, 1 mM each of dATP,dGTP, dCTP, dTTP, 0.5 units recombinant RNasin ribonuclease inhibitor(Promega) and 15 units AMV reverse transcriptase (Promega). The reactionmixture was incubated at 42 C for 60 min, heated at 95 C for 5 min thendiluted to 200 μl with sterile water. Dilutions of the reversetranscriptase reaction were then subjected to real time PCR analysis ona Taqman (PE Applied Biosystems 7700) using specific primer pairsdesigned for each gene using Primer Express software (PE AppliedBiosystems) based on the database accession number given by the genefilter manufacturer. Results were normalized to the expression of thehousekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ineach sample and are expressed as fold changes, determined by dividingthe expression value of the abnormal patient sample by the correspondingnormal patient sample.

Statistical Method for Predicting Genes Associated with Scleroderma

The statistical method for predicting genes associated with sclerodermahas been carried out by neighbor analysis and class predictor asdescribed in detail in Golub et al., 1999, which is fully incorporatedby reference herein.

Results

Gene filter microarray analysis was performed on normal and abnormalfibroblast samples from 7 scleroderma patients. The mean expressionlevel of SARP-1 cDNA for each patient sample is shown in FIG. 1 A, themedian expression is shown in FIG. 1 B. Mean and median values differsignificantly between normal and abnormal cells, the normal expressionlevel of SARP-1 being higher than the abnormal expression level. Mediansare commonly used when dealing with patient samples as they minimize theeffect of a widely variant individual within the group. Here, abnormalfibroblasts expressed significantly lower levels of SARP-1 mRNA comparedto normal fibroblasts in 5 out of 7 patients tested.

The results obtained on the microarrays were further corroborated byreal time PCR analysis of patient samples using SARP-1 specific PCRprimers. Results are shown in FIG. 2 and are expressed as the foldchange in expression level (abnormal divided by normal). In 6 out of the8 patients tested, SARP-1 was down regulated at least 2 fold in diseasedfibroblasts compared to normal fibroblasts taken from the same patient.In the 2 remaining patients the level of expression was comparable innormal and abnormal fibroblasts.

The differences in expression observed between normal and abnormalfibroblasts from the same patient are not due to differences in theculture conditions between the two populations since SARP-1 mRNAexpression in primary cultures of normal human dermal fibroblasts doesnot change significantly on passaging the cells (FIG. 3). In additionreal time RT-PCR analysis of total RNA isolated from whole biopsyspecimens of abnormal skin from scleroderma patients indicated lowerlevels of SARP-1 mRNA compared to clinically normal, age, sex andanatomical site matched control biopsies (FIG. 4).

In addition to that, the downregulation of SARP-1 was shown to have a95% probability of being associated with the progression of sclerodermausing statistical methods.

CONCLUSION

The results described above show that there is a lack of SARP-1 indiseased tissue derived from scleroderma patients as compared to healthytissue, indicating that restoring a normal level of SARP-1 may helpcuring the disease. Thus, SARP-1 may present a new medicine forachieving partial or full inhibition of the disease or of at least ofone or more symptoms of scleroderma, or for inhibition of diseaseprogression.

Example 2 Cloning of the Full cDNA Coding Sequence of Human SARP-1

Materials and Methods

Sequential BLAST searches were performed on the human dbEST (public ESTdatabase) starting with the partial coding sequence of SARP-1 (EMBLaccession number: AF017986) and relevant ESTs were retrieved usingENTREZ at www.ncbi.nlm.gov/Web/Search/index.html. The following ESTswere then assembled along with the AF017986 sequence to generate theconsensus full coding sequence of SARP-1: AW580647, AW608301, AA976403,and W92531.

The full length cDNA coding sequence of SARP-1 was then cloned byreverse transcriptase PCR using the following primers based on theconsensus sequence obtained above: SARP-1F 5′ GCC AAG CTT CCC ACG ATGCTG CAG GGC CCT (SEQ ID NO: 3) and SARP-1R 5′ GCG CTC GAG CTA GCA CTGCAG CTT GCG GAT (SEQ ID NO: 4) at 50 pmole each in a 50 μl reactionmixture containing 0.3 mM dNTPs, 1 mM MgSO₄, 5 μl of normal human dermal(foreskin) fibroblast cDNA template (prepared as described above) 5 μlof 10× Pfx amplification buffer (Life Technologies) and 1 μl of PlatinumPfx DNA polymerase (Life Technologies). The reaction mixture was heatedat 94 C for 2 min then subjected to 35 cycles of PCR as follows: 94 C 15s, 55 C for 30 s and 68 C for 1 min. The amplification products wereanalyzed on 1% agarose gels in 1×TAE buffer (Life Technologies) and PCRproducts migrating at the predicted molecular mass (906 bp) werepurified from the gel using the Wizard PCR purification kit (Promega).

100 ng of gel-purified DNA were digested with restriction enzymesHindIII and XhoI (Pharmacia), according to the manufacturer'sconditions, re-purified as described above and ligated to HindIII/XhoIdigested plasmid pcDNA3.1(+) (Invitrogen) using T4 DNA ligase (NewEngland Biolabs) according to standard molecular biology techniques.Ligation products were transformed into E. coli strain TOP 10 F′(Invitrogen) by electroporation using a Biorad Gene Pulser. Plasmid DNAwas isolated from 5 ml cultures grown up from the resultant colonies andsubjected to automated sequence analysis on an Applied Biosystems 3700sequencer using T7 and pcDNA3.1AS primers (Invitrogen) to confirm thesequence of SARP-1.

Results

In order to characterize SARP-1 activity in vitro and in vivo the fullhuman cDNA coding sequence was cloned. The partial cDNA sequence ofhuman SARP-1 (EMBL accession number AF017986) was used to identifyoverlapping ESTs in the dbEST public database using the BLAST program.The full cDNA coding sequence was assembled from the following sequenceaccession numbers: AF017986 AW580647, AW608301, AA976403, and W92531.The full-length DNA sequence and deduced amino acid sequence of SARP-1are shown in FIGS. 5A and B (A and B should be read continuously). ThecDNA coding sequence of SARP-1 was cloned by reverse transcriptase PCRusing primers which flanked the predicted start and stop codons.Sequence analysis of the resultant SARP-1 cDNA clones revealed 93%identity at the nucleotide level to the published sequence of mouseSARP-1 (Melkonyan et al., 1997) and 95% identity to the mouse sequenceat the amino acid level. The aligned human and mouse amino acidsequences of SARP-1 are depicted in FIG. 6. The predicted proteinsequence is 295 amino acids with a predicted signal peptide of 20 or 24amino acids using the SIGNALASE (Von Heijne, 1986) or SIGNALP programsrespectively, see arrows in FIG. 5 A. The amino differences between thehuman and mouse sequences are highlighted in FIG. 6 and reside in theN-terminal signal peptide (4 amino acids) and in the mature proteinsequence (2 amino acids).

SEQ ID NO: 1 of the enclosed sequence listing contains the coding strandof human SARP-1 cDNA and SEQ ID NO: 2 contains the amino acid sequenceof human SARP-1. The murine amino acid sequence is illustrated in SEQ IDNO: 5 of the enclosed sequence listing.

Example 3 N-Terminal Sequence of Human SARP-1

The predicted SARP-1 signal peptide is 20 or 24 amino acids long (seeFIG. 5A). In order to verify the correct N-terminus of mature SARP-1,recombinant SARP-1 containing a six residue histidine tag was expressedin HEK-293 cells and purified on a nickel-chelate column. The purifiedprotein migrates as a 32 kDa band in SDS-PAGE, corresponding to the massmeasured by MS, which was 34313 Da.

N-terminal sequences of the purified recombinant protein were obtainedusing an Applied Biosystems model 494 pulsed liquid phase proteinsequencer with a model 148C on-line phenylthiohydantoin amino acidanalyzer according to Maundrell et al. (1997). Briefly, purified SARP-1was digested by overnight incubation at 37° C. in 90 μl of 100 mMTris-HCl pH 8.5 containing 1 M urea, 20 mM methylamine, 1 mMdithiothreitol, and 5 μg of trypsin (Boehringer Mannheim, sequencinggrade). Peptides were separated by reverse-phase HPLC (Hewlett PackardHP1090) with a Brownlee C18 column (220×2.1 mm). Peptides were elutedwith an acetonitrile gradient (in 0.1% trifluoroacetic acid) from 0 to55% over 60 min, followed by 55-70% over 5 min. Elution fractions werecollected, and radioactive ³³P-labeled phosphopeptides identified byscintillation spectrometry were sequenced by Edman degradation using anApplied Biosystems model 494 pulsed liquid phase protein sequencer witha model 148C on-line phenylthiohydantoin amino acid analyzer.

The sequence GLFLFGQPDFSYK (residues 24-36 of SEQ ID NO:2) was obtainedby tandem mass spectrometry on a tryptic digest of the reduced andalkylated protein. Analyses were performed on a Q-TOF mass spectrometer(Micromass UK Limited, Manchester, UK) equipped with a Nano-Spray ionsource essentially as described by Cavalli et al., 2001. Briefly, onemicrogram of the purified protein was resolved by 10% SDS-PAGE. Theprotein bands were excised from the silver-stained gel and digestedin-gel with trypsin (Shevchenko et al., 1996). The extracted peptidemixture was analyzed by tandem mass spectrometric sequencing (Wilm etal., 1996) using a Q-TOF mass spectrometer (Micromass, Manchester, UK)equipped with a nano-electrospray ion source.

Results

The theoretical N-terminal sequence of immature SARP-1 is as follows:1          10         20         30         40         ... MLQGPGSLLLLFLASHCCLG SARGLFLFGQ PDFSYKRSNC KPIPANLQLC ... (residues 1-50 of SEQ IDNO:2)

The position of the 2nd predicted signal cleavage site is betweenresidue 24 and 25 (G and L).

In the sequencing experiments carried out, a considerable heterogeneityof the N-terminus of SARP-1 was found.

In one experiment, two major sequences were found, one starting withFGQPD (residues 28-32 of SEQ ID NO:2), i.e. starting at amino acid 28 ofSEQ ID NO: 2, the other one starting with LFGQPD (residues 27-32 of SEQID NO:2), i.e. starting at amino acid 27 of SEQ ID NO: 2.

However, in addition in a different purified batch 4 sequences wherefound: LFLFGQPDFS (starts at amino acid 25) (residues 25-34 of SEQ IDNO:2) FLFGQPDFS (starts at amino acid 26) (residues 26-34 of SEQ IDNO:2) LFGQPD. (starts at amino acid 27) (residues 27-32 of SEQ ID NO:2)FGQPD (starts at amino acid 28) (residues 28-32 of SEQ ID NO:2)

There is also another sequence starting at amino acid 37: RSNCKPIPAN(residues 37-46 of SEQ ID NO:2). This sequence is due to a cleavageafter a Lysine residue (tryptic like cleavage).

In addition another experiment revealed a sequence starting at position24: GLFLFGQPDFSYK (residues of 24-36 of SEQ ID NO:2)

Thus, the N-terminus seems to be very heterogeneous, which could also bedue to an endopeptidase or protease activity inherent in SARP-1. MatureSARP-1 may occur in several different variants with different N-termini.

Example 4 Transfection of SARP-1 Induces Apoptosis In Vitro

Methods

Fibroblast cell lines (mouse NIH3T3 cells and human AG1518 cells) andprimary fibroblast cultures (normal human dermal fibroblasts passage1-5) (Promocell) were transfected with plasmid pcDNA3.1 containing theSARP-1 cDNA coding sequence or pcDNA3.1 alone (empty vector-mocktransfected as a negative control) using either Geneporter 2 (GeneTherapy Systems, San Diego) or Fugene 6 (Life Technologies) transfectionreagents according to the manufacturer's recommendations. Apoptosis wasmeasured 24 h after transfection using the TiterTacs 96 well apoptosisassay kit from R&D systems and cells were enumerated using CyQuant dye(Molecular Probes) according to the manufacturer's instructions.

Results

In order to assess the effect of SARP-1 on fibroblasts, a fibroblastcell line was transfected with SARP-1 cDNA and the extent of apoptosiswas assessed. The results are shown in FIG. 7. Apoptosis in the SARP-1transfected cells was significantly reduced (P=0.0009) compared to mock(pcDNA3.1) transfected cells indicating that human SARP-1 has activitysimilar to that reported for its murine homologue. The basal level ofapoptosis was determined in non-transfected cells. Nuclease treated NIH3T3 cells served as the positive control. The column “unlabeled” showsthe background staining level, and treatment with nuclease (“nucleasetreated”) served as a positive control.

Example 5 Effect of SARP-1 on TGFβ1 Production

TGF β1 is a profibrotic cytokine which is upregulated in scleroderma andhas been previously associated with the pathogenesis of scleroderma(Kawakami et al., 1998). In order to assess whether SARP-1 downregulatesor inhibits TGFβ1 production or secretion by fibroblasts, SARP-1 can beadded to fibroblast cultures in form of purified protein. Alternatively,an expression vector comprising SARP-1 cDNA can be transfected into thecells to get a sufficient amount of SARP-1 in the cell culture. Anotherpossibility is to add medium from SARP-1 expressing cells, which may beconcentrated in order to achieve sufficient amounts of SARP-1, tofibroblast cultures. Fibroblast cell lines or primary fibroblastsderived from healthy or diseased individuals or from normal or diseasedparts of the skin of a scleroderma patient may be used for thisexperiment.

The amount of TGFβ1 can be measured by ELISA in conditioned medium fromcultured cells using the Quantikine ELISA system for TGFβ1 from R&Dsystems (cat. no. DB100).

Example 6 Effect of SARP-1 Administration on MMP-1 Activity in HumanFibroblasts In Vitro

Methods

The cDNA for human SARP-1 containing the coding sequence for a 6×His tagat the 3′ end was subcloned into the baculovirus transfer vector pDESTFastbac. C-terminal 6×His tagged SARP-1 was produced in Sf5 insect cellsinfected with recombinant baculovirus and purified by Ni-NTA affinitychromatography.

Low passage number (2-5) human fibroblasts derived from lesional ornon-lesional skin from scleroderma patients or from normal subjects weretreated in with 0, 100 or 1000 ng/ml recombinant SARP-1 for 24 h. Theconditioned medium was harvested and cell debris was removed bycentrifugation at 1200 rpm for 10 min at 4 C. MMP-1 activity wasmeasured in the undiluted culture medium using an MMP-1 fluorokine kit(purchased from R&D systems) according to the manufacturer's protocol.MMP-9 activity was also measured in the same samples.

Results

MMP-1 is responsible for the degradation of type I collagen, indicatingthat the decrease in MMP-1 activity is a contributory factor to one ofthe underlying defect in scleroderma, which involves excessive collagendeposition.

Dermal fibroblasts from scleroderma patients have decreased expressionof MMP-1 both at the mRNA level and protein level, in comparison tonormal and non-lesional dermal fibroblasts isolated from the samepatient. Although the total active MMP-1 detectable varied significantlyfrom patient to patient, SARP-1 treated fibroblasts showed higher MMP-1activity than untreated fibroblasts, both in abnormal fibroblasts fromscleroderma patients (FIG. 8 A, D, F) as well as in fibroblasts derivedfrom non-affected, healthy skin from a sclerodema patient (FIG. 8 E) andin fibroblasts from clinically normal control individuals (FIG. 8 B andC). Results shown are the means of triplicate determinations. Incontrast to MMP-1, no effect of SARP-1 was seen on MMP-9 activity (datanot shown).

Example 7 Transfection of SARP-1 cDNA Decreases the Activity of theCollagen type12 Promoter in NIH3T3 Fibroblasts

Materials and Methods

NIH3T3 cells maintained in DMEM containing 2 mM glutamine, 100 units/mlpenicillin-streptomycin and 10% FCS were plated at 50% confluency inwhite walled, transparent bottom tissue culture grade, 96 well plates(Wallac). The next day, cells were co-transfected with pcDNA3.1 SARP-1and pGL3 vector (Promega) containing the 3.5 kb collagen promoter andluciferase reporter gene (kindly provide by Dr. David Abraham, RoyalFree Hospital), using Geneporter transfection reagent according to themanufacturer's instructions. TGFβ (R&D systems, 5 ng/ml) was added 30 hafter transfection. Luciferase activity was measured directly in eachwell, 24-36 h later using the Bright-Glo assay system purchased fromPromega.

Results

In order to evaluate if there is any relationship between overexpression of SARP-1 and collagen synthesis, the effect of transfectionof SARP-1 cDNA on collagen promoter activity in NIH3T3 fibroblastsco-transfected with a Col1α2 promoter-luciferase reporter construct wasexamined.

Cotransfection of SARP-1 cDNA with a collagen promoter—luciferasereporter plasmid indicated that SARP-1 was capable of suppressing notonly the basal collagen promoter activity but also the TGFβ inducedincrease in collagen promoter activity (FIG. 9). These results suggestthat the SARP-1 gene product may be involved in the regulation of thecollagen promoter activity in vitro either directly or indirectlythrough SARP-1 mediated signaling events.

Another way of testing the effect of SARP-1 on collagen depositionand/or synthesis and/or secretion in fibroblasts is adding purifiedSARP-1 to fibroblast cultures. Alternatively, an expression vectorcomprising SARP-1 cDNA can be transfected into the fibroblasts in orderto achieve a sufficient concentration of SARP-1 in the cell culture.

Another possibility is to add medium from SARP-1 expressing cells, whichmay be concentrated in order to achieve sufficient concentrations ofSARP-1, to fibroblast cultures. Fibroblast cell lines or primaryfibroblasts derived from healthy or diseased individuals or from normalor diseased parts of the skin of a scleroderma patient may be used forthis experiment.

Collagen synthesis can be measured in vitro in cultured humanfibroblasts using a capture ELISA system (antibodies purchased fromSouthern Biotechnology Associates Inc) as described by Shi-wen et al.,1997.

Example 8 Human Dermal Fibroblasts Overexpressing SARP-1 or Treated withRecombinant Human SARP-1 Show Altered mRNA expression of GenesAssociated with the Pathology of Scleroderma

Materials and Methods

Normal human dermal (foreskin) fibroblasts were maintained in fibroblastculture medium (Promocell). Fibroblasts were treated with recombinanthuman SARP-1 (100 ng/ml) for 4 or 24 h in the presence and absence ofhuman TGFβ1. Cells were harvested after the treatment period and totalRNA was isolated using the Trizol™ reagent (Life Technologies) accordingto the manufacturer's instructions. A 32P-dATP labelled cDNA probe wasprepared from the RNA as described above and hybridized to an R&D sytemshuman cytokine gene filter array and processed as described above.

Results

Although SARP-1 was initially described as an apoptosis related protein,its precise function is unknown. In order to gain an insight into therole of SARP-1 in scleroderma, the effect of treatment of TGFβpretreated human dermal fibroblasts, which mimics sclerodermaphenotyope, with SARP-1 on fibroblast gene expression was measured.

Table IV shows mRNAs affected by treatment of fibroblasts withrecombinant SARP-1 after prior stimulation for 4 h with TGFα, which wasused to mimic the scleroderma phenotype. Cells were treated with SARP-1for 24 h. Under these conditions, TGFα and FGF 5 were down regulated.Decreased expression of endoglin, a TGFβ1 binding protein, and increasedexpression of furin, a protease associated with the activation of matrixmetalloproteases, were observed. Another mRNA which was down regulatedwas TARC. This chemokine is associated with the recruitment of skinhoming T cells to inflammatory sites. It is therefore possible thatSARP-1 administration may have anti-inflammatory effects. Upregulationof TNFR1 subunit mRNA was also observed. The significance of thisobservation is unclear as there appears to be much discrepancy in thescientific literature as to whether TNF has a pathogenic or protectiverole in scleroderma.

In a separate experiment (not shown), regulation of genes in fibroblastsfrom scleroderma patients had been analyzed in comparison to healthycontrol fibroblasts. Interestingly, the regulated genes indicated inTable IV had been found to be regulated in scleroderma samples. Thosegenes found up-regulated in scleroderma fibroblasts were down-regulatedafter SARP-1 expression in the above model, and those genes founddown-regulated were found up-regulated after SARP-1 expression in theabove model, further indicating that SARP-1 may have a beneficial effectin treatment of scleroderma. TABLE IV TGFβ + SARP-1 Ratio * TGFβ treatedtreated Gene −1.6 1346 830 FGF-5 −2.7 788 297 endoglin 3.3 157 515 TNFRI3.3 128 428 furin −4.6 275 60 TGFα −4.7 164 34 TARC* Ratio SARP-1/mock represents the ratio TGFα + SARP-1 treated/TGFαalone.

Results shown are based on two independent experiments

Example 9 Assessment of SARP-1 Effects In Vivo in a Murine Model ofDisease

The bleomycin-induced lung injury is an accepted model for scleroderma.The disease is induced by intra-tracheal administration of bleomycin(Hattori et al., 2000).

Treated mice develop an inflammatory response in the lungs (maximumaround day 7) followed by fibrosis that peaks around day 14post-induction. This model was used to assess the effect of SARP-1administration in vivo on the development of lung fibrosis. Acell-delivery system (NIH3T3 cells transfected with full length SARP-1cDNA), was used to generate SARP-1 in vivo.

Methods

Animal Treatments.

Lung fibrosis was induced in mice (20-25 g) by intra-trachealadministration of bleomycin (0.075 IU) in saline on day 1. Mice weredivided into 4 separate groups of 10 animals. One group received an i.p.injection of 10⁶ NIH3T3 cells transfected with SARP-1/pcDNA3.1. A secondgroup received 106 mock transfected (pcDNA3.1 vector) NIH3T3 cells. Thethird group received 250 μg of anti-TGFβ antibody (Anti Pan TGFβ: Sigmacat. no. T9429) and mice in the final group received saline. All micewere sacrificed at day 14 and lungs were formalin fixed and paraffinembedded for histology. Lung sections were stained with tri-chrome orpicro sirius red for collagen (Bancroft J. D. and Stevens A. Theory andPractice of Histological Techniques) and the extent of fibrosis/lung wasscored.

Transfection of NIH3T3 Cells

NIH 3T3 cells were transfected with SARP-1/pcDNA3.1 or pcDNA3.1 usingthe Geneporter 2 reagent as described previously. Twenty four hoursafter transfection the culture medium was removed and cells were treatedwith phosphate buffered saline (PBS) containing 1 mM EDTA for 5 min at37 C. Cells were gently detached with a cell scraper (Costar),centrifuged for 5 min at 4 C to pellet the cells, and resuspended ininjection grade saline at a concentration of 10⁷ cells/ml.

Results

SARP-1 protects against bleomycin induced lung fibrosis.

Animals treated with bleomycin alone, or with mock transfected NIH3T3cells developed significant lung fibrosis (not shown). TGFβ1 is thoughtto be one of the key agents responsible for the pathological changesleading to fibrosis. An anti TGFβ1 polyclonal antibody has previouslybeen shown to protect against the development of lung fibrosis whenadministered prophylactically (Yamamoto et al., 1999) and also reducesskin fibrosis in the GvHD model of scleroderma (McCormick et al., 1999).Therefore, this antibody was used as a positive control in theseexperiments. Anti-TGFβ treated mice did not develop extensive fibrosisof the lungs. However mice treated with SARP-1 transfected cells had amarkedly reduced number of fibrotic lesions in the lung when compared tomice treated with bleomycin alone or with mock transfected cells. Theeffect was comparable or even better than that seen with the anti TGFβtreatment (not shown).

Histological analysis of the lungs shows that the SARP-1 treated animalshad close to normal morphology of the lungs with little inflammatoryinfiltrate and mostly normal alveolar architecture. This morphologyresembles the morphology of the anti-TGFβ-treated animals and is insharp contrast to the abnormal morphology seen in the lungs from mockand untreated animals. In the latter 2 groups, the alveolar spaces arefilled by cellular infiltrates and collagen deposition (not shown).

The amounts of fibrotic lesions in lungs of the different experimentalgroups are depicted in FIG. 10. SARP-1 treated mice containedsignificantly fewer fibrotic lesions as compared to saline or anti-TGFβtreated mice.

Administration of bleomycin can lead to increased mortality. In additionto the protective effects of SARP-1 administration against fibrosis itwas also noted that larger numbers of mice survived the treatment (8/10mice) compared to the saline treated mice (6/10), anti TGFβ treated mice(4/10) and the mock treated mice (3/10). Therefore, SARP-1 treatmenteven protects from death in the lung fibrosis model, indicating thatSARP-1 treatment is clearly superior to anti TGFβ treatment in thisexperimental setting.

In summary, in this established in vivo model of scleroderma, abeneficial effect of SARP-1 has been demonstrated.

Further in vivo models can be used to test the effect of SARP-1administration, such as a murine sclerodermatous graft-vs-host disease(Scl GVHD) model for scleroderma. This model reproduces importantfeatures of scleroderma including skin thickening, lung fibrosis, andup-regulation of cutaneous collagen mRNA, which is preceded by monocyteinfiltration and the up-regulation of cutaneous TGF-beta1 mRNA. Themodel is described in detail by (McCormick L L et al., 1999).

Example 10 Systemic Delivery of SARP-1 by Intramuscular Injection ofExpression Plasmid DNA

To demonstrate the role of SARP-1 as a protective factor in scleroderma,mice are transfected in vivo with a plasmid encoding the murine SARP-1cDNA and are compared to mice transfected with an empty plasmid ascontrol.

Plasmids are injected in both tibial cranial muscles of the anesthetisedmouse as previously described (Dimmeler et al., 1997; Mir et al., 1999).Briefly, transcutaneous electric pulses (8 square wave electric pulsesof 200 V/cm, 20 msec duration at 2 Hz) are delivered by a PS-15electropulsator using two stainless steel plate electrodes placed 4.2 to5.3 mm apart, at each side of the leg.

Then, bleomycin-induced lung fibrosis is as explained in the previousexample and development of disease is observed in both SARP-1transfected and mock transfected animals.

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1. A method for treating liver cirrhosis or interstitial pulmonaryfibrosis, comprising administering to a patient in need thereof apolypeptide that binds to Wnt protein to competitively inhibit thebinding of Wnt protein to its receptor and to treat liver cirrhosis orinterstitial pulmonary fibrosis, wherein said polypeptide is selectedfrom the group consisting of: a) mature secreted apoptosis-relatedprotein 1 (SARP-1) comprising at least 90% sequence identity to aminoacids 21 to 29 of SEQ ID NO:2; b) a fragment of a) comprising at leastthe cysteine rich frizzled domain thereof; c) a polypeptide comprisingSEQ ID NO:2; d) a polypeptide comprising amino acids 21 to 295 of SEQ IDNO:2; e) a polypeptide comprising amino acids 24 to 295 of SEQ ID NO:2;f) a polypeptide comprising amino acids 25 to 295 of SEQ ID NO:2; g) apolypeptide comprising amino acids 26 to 295 of SEQ ID NO:2; h) apolypeptide comprising amino acids 27 to 295 of SEQ ID NO:2; i) apolypeptide comprising amino acids 28 to 295 of SEQ ID NO:2; j) apolypeptide comprising amino acids 37 to 295 of SEQ ID NO:2; k) a muteinof any of (a) to (j), wherein the amino acid sequence of said mutein hasat least 90% sequence identity to at least one of the sequences in (a)to (j); l) the mutein of (k), wherein any changes in the amino acidsequence are conservative amino acid substitutions to the amino acidsequences in (k) m) a salt, an isoform of (a)-(l), or a fusion proteinof any of (a) to (l).
 2. The method of claim 1, wherein the polypeptideis glycosylated.
 3. The method of claim 1, wherein said (m) fusedprotein is a fusion between said polypeptide and an immunoglobulin. 4.The method of claim 1, wherein said polypeptide is present in apharmaceutical composition that further comprises an interferon.
 5. Themethod of claim 4, wherein the interferon is interferon-β.
 6. The methodof claim 1, wherein said polypeptide is administered systemically. 7.The method of claim 1, wherein said polypeptide is administered byintramuscular injection.
 8. The method of claim 1, wherein saidpolypeptide is administered by inhalation.
 9. The method of claim 1,wherein said polypeptide is administered subcutaneously.